Over the last 25 years, remarkable progress has been made in our basic knowledge and treatment of HIV-1, Despite such achievements however, AIDS remains a global pandemic that results in millions of fatalities each year. We must therefore continue to expand our knowledge of the virus and its interaction with the host in order to generate better options for both effective anti-HIV therapies and successful vaccine development. The human protein BST-2 was recently determined to possess the unique ability to restrict the egress of HIV-1 and other enveloped viruses. Coincident with this discovery was the finding that the HIV-1 protein Vpu counteracts BST-2, thereby allowing viral progeny to readily escape from infected cells. The goal of this application is to expand the current knowledge base by achieving a comprehensive understanding of how HIV Vpu subjugates the novel innate immune molecule BST-2, and by identifying other proteins involved in the host restriction and/or viral antagonism. The Specific Aims and Research Plan of this application are: S.A.1: To map the critical residues of Vpu outside of the TrCP-binding domain that are necessary for BST-2 interaction and degradation. This aim will be accomplished by exploiting the serendipitous discovery of a naturally occurring HIV-1 subtype C Vpu variant that has lost the ability to downregulate or interact with BST-2. A sequence comparison between the active subtype B Vpu and the inactive subtype C Vpu proteins has revealed potential regions and residues of interest. These findings will be used to direct the design and generation of Vpu chimeras and point mutants that will then be assessed for their impact upon BST-2 binding and degradation. S.A.2: To characterize the mechanism of Vpu-mediated degradation of BST-2. This aim will be achieved by determining the role that ubiquitin plays in BST-2 degradation, the timing and cellular location of Vpu action upon BST-2, the forms of BST-2 that are degraded in the presence of Vpu, and the requirement for ESCRT components in these processes. S.A.3: To identify BST-2 binding partners that are involved in HIV virion tethering or that act as adapters participating in Vpu-mediated BST-2 degradation. Three separate protein- interaction screens will be employed to accomplish this aim, including a modified yeast 2-hybrid approach, a phage display screen, and a proteomics-based co-immunoprecipitation strategy. All proteins that are found to interact with BST-2 will then be evaluated for their impact upon both viral egress and Vpu-dependent BST-2 degradation. Numerous publications have demonstrated that the ability of Vpu to enhance viral release is an important component of HIV pathogenicity, so the comprehensive analysis of this virus/host interaction proposed in this application will provide important insights that could lead to novel antiviral targets. The genetic and functional analyses proposed in this application will also provide valuable perspectives regarding the antiviral function of the newly identified immune modulator known as BST-2.